Biological assays are mainly based upon interaction specificity between two biological molecules such two strands of nucleic acid molecules, an antigen and an antibody or a ligand and its receptor. The present challenge of biological assays is to perform simultaneously the multiple detection of molecules present in a sample. Miniaturisation and development of arrays upon the surface of “biochips” are tools that allow multiplex reactions in a microscopic format, said detection being made with a limited volume of sample for the screening and/or the identification of multiple possible target compounds. These arrays are formed of discrete regions, containing a specific capture molecule used for the binding of the target compound. These discrete regions, as small as a few micrometers, allow the fixation of several thousands capture molecules per cm2 surface (WO 95/11995).
However, the detection of bounded target compounds is difficult, since their amount is very small due to said miniaturisation (few fentomoles or even few attomoles). Therefore, only extremely sensitive methods are adequate for such detection.
It has been proposed a labelling of a target compound like DNA with fluorescent molecules after their possible genetic amplification. When an RNA molecule has to be detected, it is first transformed into a cDNA, before its possible amplification. If direct labelling of the target compound is not possible, a double reaction (sandwich reaction) can be performed. However, the amount of fluorescent molecules is so low that it is necessary to develop specific array scanners for the detection and/or the quantification of the bounded compound upon the “hybridisation chips”. Said expensive specific scanners comprise a laser scanner for excitation of the fluorescent molecules, a pinhole for decreasing the noise fluorescent background, and a photomultiplier for increasing the sensitivity of the detection,
Alternative detection methods that present a high sensitivity are described in the documents U.S. Pat. No. 5,821,060 and WO95/04160 and are based upon the detection using expensive devices such as mass spectrometers It has also been proposed methods based upon the precipitation of specific products resulting of a calorimetric labelling (U.S. Pat. Nos. 5,270,167, 4,731,325, EP-A-0301141) or the result of an enzymatic activity (EP-A-0393868, WO 86/02733, EP-A-0063810). However, said methods are either characterized by a low sensitivity or are not adequate for the detection of a target compound upon “hybridisation chips”, because the precipitate will occur at a certain distance of the reaction binding and its location can not be easily correlated with a specific bounded target compound. In addition, the density of the precipitate of such enzymatic reactions is not enough opaque for allowing a detection by light absorption.
It has also been proposed to improve the detection by fixing a soluble product obtained from the enzymatic reaction with a metal before its precipitation. However, as the result of said enzymatic reaction is a soluble product, there is no correlation between the location of the precipitate and the detection of a specific bounded target compound.